1. Field of the Invention
The present invention relates to biotechnology and, more specifically, to a method for producing amino acids by fermentation using amino acid producing bacterium belonging to the genus Escherichia capable of utilizing sucrose a sole carbon source.
2. Description of the Related Art
Sucrose and sucrose containing substrates (e.g. molasses) are often used as a starting point for the microbial production of commercial products such as amino acids, vitamins and organic acids. The process for production of amino acids from carbohydrates strives to maximize the efficiency with which the carbon skeleton of carbohydrate is converted into desired product.
The majority of sucrose-positive bacteria take up and phosphorylate sucrose by a phosphoenol pyruvate-dependent, sucrose-6-phosphotransferase system (sucrose PTS) to yield intracellular sucrose-6-phosphate. This phosphate is hydrolyzed by a sucrose-6-phosphate hydrolase (invertase or sucrase) into D-glucose 6-phosphate and D-fructose, which is itself phosphorylated by an ATP-D-fructose-6-phosphate phosphotransferase (fructokinase). Such systems and metabolic pathways have been described at the molecular level for the gram-positive bacteria Bacillus subtilis and Streptococcus mutans (Debarbouille et al., 1991. Res. Microbiol., 142: 757-764; Sato et al., 1989. J. Bacteriol., 171: 263-271) and for gram-negative bacteria. Further plasmid-coded pUR400 system from enteric bacteria has been reported (Aulkemeyer et al., (1991) Mol. Microbiol., 5: 2913-2922; Schmid et al., 1988. Mol. Microbiol., 2: 1-8; Schmid et al., 1991. Mol. Microbiol., 5: 941-950).
Although about 50% of wild-type isolates of Escherichia coli are sucrose positive, the laboratory E. coli strains such as E. coli K-12, E. coli B, E. coli C which are now used in the breeding of the industrially important producing strains cannot utilize sucrose. However, this property may be easily provided to these strains by introducing sucrose utilization genes from sucrose-positive E. coli or Salmonella strains using conjugation, transduction or cloning procedures (Wohlhieter et al., 1975. J. Bacteriol., 122:401-406; Parsell and Smith, 1975. J. Gen. Microbiol., 87: 129-137; Alaeddinoglu and Charles, 1979. J. Gen. Microbiol., 110:47-59; Livshits et al., 1982. In: Metabolic plasmids. P.132-134; Garsia, 1985. Mol. Gen. Genet., 201:575-577; U.S. Pat. No. 5,175,107).
Phosphoenol pyruvate (PEP) is one of the major building blocks in several biosynthetic pathways. PEP is combined with carbon dioxide to produce oxaloacetic acid. Oxaloacetic acid serves as the carbon skeleton for aspartic acid, asparagine, threonine, isoleucine, methionine and lysine. Besides, an equimolar amount PEP is condensed with erythrose-4-phosphate to form 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP), the first intermediate of the common segment of the aromatic pathway. From this metabolic route such commercially important amino acids as tryptophan, phenylalanine and tyrosine can be obtained. The yield of these metabolites may be limited by PEP availability.
During glycolysis four moles of PEP are produced from two mole of glucose, and half of the PEP is obligatory consumed to provide energy for glucose uptake. In the case of sucrose internalization two moles of hexose (glucose and fructose) arising from one mole of sucrose also produce four moles of PEP, but only one mole is consumed for the sucrose transport, thus increasing 1.5 times the amount of PEP available as a source of carbon skeletons for biosynthesis. Therefore, it is possible to improve the amino acid yield by providing the E. coli amino acid producing strains with the ability to utilize sucrose, and using sucrose or sucrose containing substrates as a carbon source.
Known in the present state of the art is the threonine producing strain VKPM B-3996 based on E. coli K-12 capable of sucrose utilization (U.S. Pat. No. 5,705,371). The restriction and sequence analysis of the cloned sucrose genes from the VKPM B-3996 strain showed that they are almost identical to those of pUR400 (accession numbers: EMBL X61005; EMBL X67750, GB M38416) encoding PTS sucrose transport and metabolism (Lengeler et al., 1982. J. Bacteriol., 151:468-471; Schmid et al., 1988, Mol. Microbiol., 2:1-8; Schmid et al., 1991, Mol. Microbiol., 5:941-950).
A chromosomally encoded, non-PTS metabolic pathway for sucrose utilization was also found in Escherichia coil (Bockmann et al., 1992, Mol. Gen. Genet., 235:22-32). The pathway involves a proton symport transport system (LacY type permease), an invertase, a fructokinase, and a sucrose-specific repressor. By using this non-PTS metabolic pathway the output of an amino acid derived from a PEP precursor could be further increased because sucrose transport into the cells would not be coupled to PEP. However this approach was never used before for the amino acid producing strain improvement.